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SAM 121.05

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 121

Supplemental Assay Method for Titration of Porcine Rotavirus in 

Modified-live Vaccines

Date: October 21, 2014 Approved, Pending Standard Requirement

Number: SAM 121.05

Supersedes: SAM 121.04, September 9, 2011

Standard Requirement:

Contact: Alethea M. Fry, (515) 337-7200

Peg A. Patterson

Approvals: /s/Geetha B. Srinivas Date: 23Dec14

Geetha B. Srinivas, Section Leader

Virology

/s/Byron E. Rippke Date: 29Dec14

Byron E. Rippke, Director

Policy, Evaluation, and Licensing

Center for Veterinary Biologics

/s/Rebecca L.W. Hyde Date: 29Dec14

Rebecca L.W. Hyde, Section Leader

Quality Management

Center for Veterinary Biologics

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by USDA 

and does not imply its approval to the exclusion of other products that may be suitable.
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Supplemental Assay Method for the Titration of Porcine Rotavirus in Modified-live Vaccines

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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Report of Test Results

7. References

8. Summary of Revisions

Appendix
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Supplemental Assay Method for the Titration of Porcine Rotavirus in Modified-live Vaccines

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1. Introduction

This Supplemental Assay Method (SAM) describes an in vitro assay method which utilizes 

cytopathic effect (CPE) in a cell culture system or an indirect fluorescent antibody (IFA) technique 

for determining the Group A porcine rotavirus (PROTA) content of modified-live vaccines.

Note: For this SAM, the dilution terminology of 1:10, 1:20, etc., specifies 1 part plus 9 parts 

(liquid), 1 part plus 19 parts, etc.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 36°± 2°C, 5% ± 1% CO2, high-humidity incubator (Model 3158, Forma 

Scientific Inc.)

2.1.2 Water bath

2.1.3 Microscope, inverted light (Model CK, Olympus America Inc.)

2.1.4 Microscope, ultraviolet (UV) light (Model BH2, Olympus America Inc.)

2.1.5 Vortex mixer (Vortex-2 Genie, Model G-560, Scientific Industries Inc.)

2.1.6 Micropipettors: 200-μL and 1000-μL single channel; 300-μL x 12-channel

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below. All 

reagents and supplies must be sterile.

2.2.1 PROTA Reference Viruses

1. Serotype 4 (Gottfried strain)

2. Serotype 5 (OSU strain)

2.2.2 Rhesus monkey kidney cells (MA-104) free of extraneous agents as tested 

by title 9, Code of Federal Regulations (9 CFR) (available from the Center for 

Veterinary Biologics (CVB)).
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2.2.3 Minimum essential medium (MEM) (National Centers for Animal 

Health (NCAH) Media #20030)

1. 9.61 g MEM with Earles salts without bicarbonate

2. 1.1 g sodium bicarbonate (NaHCO3)

3. Q.S. to 1000 mL with deionized water (DI); adjust pH to 6.8-6.9 with 

2N hydrochloric acid (HCl).

4. Sterilize through a 0.22-μm filter.

5. Aseptically add:

a. 10 mL L-glutamine (200 mM)

b. 50 μg/mL gentamicin sulfate

6. Store at 2°- 7°C.

2.2.4 Growth Medium

1. 930 mL of MEM

2. Aseptically add 70 mL of gamma-irradiated fetal bovine serum (FBS).

3. Store at 2°- 7°C.

2.2.5 Diluent Medium

1. 100 mL MEM

2. 83.3 μL pancreatin (4XNF 10X)

3. Store at 2°- 7°C. 

2.2.6 Anti-PROTA monoclonal antibody (MAb)

1. MAb against Serotype 4 (Gottfried strain)

2. MAb against Serotype 5 (OSU strain)

2.2.7 Rabbit anti-mouse fluorescein isothiocyanate-labeled conjugate (Rabbit 

Anti-mouse Conjugate)
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2.2.8 Anti-PROTA serum against OSU strain

2.2.9 Anti-PROTA serum against Gottfried strains

2.2.10 0.01 M Phosphate buffered saline (PBS) (NCAH Media #30054)

1. 1.19 g sodium phosphate, dibasic, anhydrous (Na2HPO4)

2. 0.22 g sodium phosphate, monobasic, monohydrate (NaH2PO4•H2O)

3. 8.5 g sodium chloride (NaCl)

4. Q.S. to 1000 mL with DI.

5. Adjust pH to 7.2-7.6 with 0.1 N sodium hydroxide (NaOH) or 2.0 N 

HCl.

6. Sterilize by autoclaving at 15 psi, 121°± 2°C for 35 ± 5 minutes.

7. Store at 2°- 7°C.

2.2.11 80% Acetone

1. 80 mL acetone

2. 20 mL DI

3. Store at room temperature. 

2.2.12 Cell culture plates, 96-well

2.2.13 Polystyrene tubes, 12 x 75-mm

2.2.14 Graduated cylinders, 25-mL, 50-mL, 100-mL, and 250-mL, sterile

2.2.15 Pipette

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have training in the immunological basis of cell culture techniques, the 

principles of IFA, and aseptic techniques.
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3.2 Preparation of equipment/instrumentation

On the day of test initiation, set a water bath at 36°± 2°C.

3.3 Preparation of reagents/control procedures

3.3.1 MA-104 Plates

Two days prior to test initiation, seed 96-well cell culture plates with MA-104 

cells, in Growth Medium, at a cell count that will produce a monolayer after 2 

days of incubation at 36°± 2°C in a CO2 incubator. These become the MA-104 

Plates. One test vaccine or 1 of the 2 PROTA Reference Viruses can be tested on 

each MA-104 Plate. Growth Medium is changed if excess acidity of the medium 

is observed or cells are not confluent after incubation.

3.3.2 Pipette 2.0 mL of Dilution Medium into 2, 12 x 75-mm polystyrene tubes. 

Label each tube with the appropriate Reference Virus.

1. Transfer 500 μL of each Reference Virus to the corresponding labeled 

tube; mix by vortexing. Discard pipette tip. This is a 1:5 dilution of the 

Reference Virus.

2. Transfer 1.0 mL of each 1:5 Reference Virus to 2, 12 x 75-mm 

polystyrene tubes labeled 10-1for each Reference Virus (total of 4 tubes).

3. Pipette 1.0 mL/tube of a specific Anti-PROTA serum to separate 10-1

labeled tubes of each Reference Virus. This becomes the Reference VirusSpecific Antiserum Mixture. Repeat for the other specific Anti-PROTA serum 

(see Appendix).

4. Mix with the vortex mixer; incubate for 2 hours ± 10 minutes at room 

temperature.

5. Pipette 1.8 mL/tube of Diluent Medium into 2 sets of 12 x 75-mm 

polystyrene tubes labeled 10-2through 10-8for each Reference Virus-Specific 

Antiserum Mixture.

6. Prepare vaccine tenfold dilutions of each PROTA Reference VirusSpecific Antiserum Mixture. Transfer 200 μL from each Reference VirusSpecific Antiserum Mixture tube to the corresponding labeled 10-2

tube. Mix 

with the vortex mixer. Discard pipette tip.

7. Repeat Step 6 for the remaining tubes, transferring 200 μL sequentially 

from the previous dilution to the next dilution, until the dilution sequence is 

completed.
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3.3.3 Working Anti-PROTA MAb

On the day of MA-104 plate examination, if an IFA test is to be conducted, dilute the 

appropriate Anti-PROTA MAb in PBS, according to the CVB supplied Reagent Data 

Sheet or as determined for that specific MAb.

3.3.4 Working Rabbit Anti-mouse Conjugate

On the day of MA-104 plate examination, if an IFA test is to be conducted, dilute the 

Rabbit Anti-mouse Conjugate in PBS, according to the manufacturer’s 

recommendations.

3.4 Preparation of the sample

3.4.1 Conduct the initial test of a Test Vaccine with a single vial (a single sample 

from 1 vial). On the day of test initiation, remove the seal and stopper from both the 

Test Vaccine bottle and the bottle containing the accompanying diluent. Measure the 

diluent into a sterile graduated cylinder according to the number of doses indicated on 

the manufacturer’s instructions (e.g., for a 50-dose container of 2-mL-per-dose, 

reconstitute with 100 mL of diluent), and aseptically pour the diluent into the 

lyophilized bottle of vaccine. Mix by vortexing.

3.4.2 Neutralize Test Vaccines containing multiple PROTA and make vaccine 

tenfold dilutions similar to the methods described in Section 3.3.2 (see Appendix).

4. Performance of the Test

4.1 On the day of test initiation, decant Growth Medium from the MA-104 Plates.

4.2 Add 200 μL/well Diluent Medium to the MA-104 Plates. Decant the Diluent 

Medium.

4.3 Again, add 200 μL/well Diluent Medium to the MA-104 Plates and incubate for 60 ± 

10 minutes at 36°± 2°C in a CO2 incubator. Decant the Diluent Medium.

4.4 Inoculate 200 μL/well of each dilution of the Test Vaccine-Specific Antiserum 

Mixture and each Reference Virus-Specific Antiserum Mixture into a minimum of 5 

wells/dilution of an MA-104 Plate.

4.5 Add 200 μL/well Diluent Medium to a minimum of 5 wells on each MA-104 plate, to 

serve as uninoculated cell controls.

4.6 Incubate the MA-104 Plates for 5 days ± 1 day postinoculation at 36°± 2°C in a 

CO2 incubator.
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4.7 CPE counting is the primary method of determining the log10 50% tissue culture 

infective dose (TCID50).

4.7.1 After 5 days ± 1 day postinoculation, examine the wells with an inverted light 

microscope. The CPE of PROTA is visible as cell death in the cell monolayer.

4.7.2 Record the number of wells/dilution showing any characteristic PROTA CPE 

for each Test Vaccine-Specific Antiserum Mixture and Reference Virus-Specific 

Antiserum Mixture.

4.7.3 Calculate the TCID50 of the Test Vaccine-Specific Antiserum Mixture and 

each Reference Virus-Specific Antiserum Mixture using the method of SpearmanKärber as modified by Finney.

Example:

100 dilution of the Test Vaccine = 5 of 5 wells CPE Pos

10-1dilution of the Test Vaccine = 5 of 5 wells CPE Pos

10-2dilution of the Test Vaccine = 3 of 5 wells CPE Pos

10-3dilution of the Test Vaccine = 0 of 5 wells CPE Pos

Test Vaccine titer = (X - d/2 + [d • S]) where:

X = Log10 of lowest dilution (=0)

d = Log10 of dilution factor (=1)

S = Sum of proportion of CPE positive 

5 5 3 0

5 = 1.0 + 5 = 1.0 + 5 = 0.6 + 5 = 0 = 2.6

Titer = (0 – 0.5 + [1 • 2.6]) = 2.1

Adjust the titer to the recommended Test Vaccine dose as follows:

1. Divide the Test Vaccine Dose by the Inoculation Dose

Test Vaccine Dose = manufacturer’s recommended vaccination dose (for 

this test vaccine, the recommended dose is 2 mL)

Inoculation Dose = amount of diluted Test Vaccine added to each well of 

the Test Plate (for this test vaccine, the inoculation dose is 0.2 mL)

2 mL dose

 0.2 mL = 10
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2. Calculate log10 of value in A and add it to the Test Vaccine titer as 

illustrated below:

Log of 10 = 1.0

Test Vaccine titer = 2.1 + 1.0 = 3.1

Therefore the titer of the Test Vaccine is 103.1TCID50/1 mL.

4.8 Certain strains of PROTA may not exhibit pronounced CPE. An IFA may be 

conducted to determine the titer:

4.8.1 Decant Growth Media from the MA-104 Plates.

4.8.2 Rinse the MA-104 Plates with PBS and incubate at room temperature for 5 ± 

2 minutes. Decant the PBS.

4.8.3 Fill the wells with 80% acetone and incubate at room temperature for 15 ± 5 

minutes.

4.8.4 Decant the 80% acetone from the MA-104 Plates and air dry at room 

temperature.

4.8.5 Pipette 35 μL of the Working Anti-PROTA MAb into all wells and incubate 

for 45 ± 15 minutes at room temperature.

4.8.6 Decant the Working Anti-PROTA MAb. Fill the wells completely with 

PBS and incubate at room temperature for 5 ± 2 minutes. Decant the PBS.

4.8.7 Repeat for a total of 2 washes.

4.8.8 Gently tap the MA-104 Plates onto paper towels to remove excess liquid.

4.8.9 Pipette 35 μL of the Working Rabbit Anti-mouse Conjugate into all wells and 

incubate for 40 ± 10 minutes at room temperature.

4.8.10 Repeat Steps 6 through 8.

4.8.11 Fill the wells completely with distilled water; decant. Allow to air dry or dry 

at 36°± 2°C.

4.8.12 Examine the MA-104 Plates with a UV-light microscope at 100 to 200X 

magnification.
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4.8.13 A well is considered positive if typical cytoplasmic, apple-green 

fluorescence is observed.

4.8.14 Record and calculate as in Sections 4.7.2 and 4.7.3.

5. Interpretation of the Test Results

5.1 The test is not a valid test if CPE, fluorescence, or bacterial/fungal contamination is 

observed in any of the uninoculated control wells.

5.2 An Anti-PROTA serum must neutralize the corresponding homologous Reference 

Virus.

5.3 The calculated titer of each Reference Virus with heterologous antiserum must 

fall within plus or minus 2 standard deviations (± 2 SD) of its mean titer, as established 

from a minimum of 10 previously determined titers.

5.4 If the validity requirements are not met, then the assay is considered a NO TEST

and can be retested without prejudice.

5.5 If the validity requirements are met and the titer of the Test Vaccine is greater than or 

equal to the titer contained in the Animal and Plant Health Inspection Service (APHIS) filed 

Outline of Production or Special Outline, the Test Vaccine is considered SATISFACTORY.

5.6 If the validity requirements are met but the titer of the Test Vaccine is lower than the 

required minimum contained in the APHIS filed Outline of Production or Special Outline, 

the Test Vaccine may be retested in accordance with the 9 CFR 113.8.

6. Report of Test Results

Record all test results on the test record. The titer of the test vaccine is reported as the log10

TCID50 per dose.

7. References

7.1 Title 9, Code of Federal Regulations, U.S. Government Printing Office, Washington, 

DC.

7.2 Conrath TB. Handbook of Microtiter Procedures. Clinical and Research Applications 

Laboratory, Alexandria, Virginia: Cooke Engineering Co., 1972.
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7.3 Finney DJ. Statistical Method in Biological Assay, 3rd ed. London: Charles Griffin 

and Co., 1978, pp. 394-401.

7.4 Rose NR, Friedman H, Fahey JL, eds. Neutralization Assays. In: Manual of 

Clinical Laboratory Immunology. Washington, DC: ASM 1986.

8. Summary of Revisions

Version .05

 The Contact information has been updated; however, the Virology Section has elected 

to keep the same next review date for the document.

Version .04

 The phrase "available from the Center for Veterinary Biologics/CVB" has been 

removed from the document as these reagents are no longer supplied by the CVB.

Version .03

 The document number has been changed from VIRSAM0121 to SAM 121.

 The Contact information has been updated.

Version .02

This document was revised to clarify the practices currently in use at the Center for Veterinary 

Biologics and to provide additional detail. While no significant changes were made that impact the 

outcome of the test, the following changes were made to the document:

 2.1.7 has been deleted.

 2.2.3.2 The amount of sodium bicarbonate (NaHCO3) has been changed from 2.2 g 

to 1.1 g.



 2.2.3.5 Penicillin, streptomycin and amphotericin B have been removed.

 2.8 Anti-PROTA serums have been separated.

 2.2.15 Pipette has been added.

 3.3.2.2 “Repetitive syringe” has been deleted and “pipette” has been added.
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 4.4 “Specific Antiserum Mixture” has been added to Test Vaccine.

 4.7.3 Additional steps have been added to clarify the titer calculations by SpearmanKärber formula.

 4.8.6 “Decant the working Anti-PROTA(MAb)” has been added.

 4.8.11 “Fill the wells completely with DI” has been added.

 5.3 “With heterologous antiserum” has been added after Reference Virus.

 The refrigeration temperatures have been changed from 4° ± 2°C to 2°- 7°C. This reflects 

the parameters established and monitored by the Rees system. 

 “Test Serial” has been changed to “Test Vaccine” throughout the document.

 “Reference and Reagent Sheet” has been changed to “Reagent Data Sheet” 

throughout the document.
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Appendix

Gottfried RV* (1:5): 1 mL + 1 mL Anti-Gottfried AS

1 mL + 1 mL Anti-OSU AS

OSU RV* (1:5): 1 mL + 1 mL Anti-Gottfried AS

1 mL + 1 mL Anti-OSU AS

Test Vaccine (1:5): 1 mL + 1 mL Anti-Gottfried AS

1 mL + 1 mL Anti-OSU AS

* RV = Reference Virus

 AS = Anti-PROTA serum